DESCRIPTION this project is to analyze the basic mechanisms by which cell injury occurs in anoxic cardiac muscle. The project will use anoxic guinea-pig ventricular myocytes as the experimental model, and is designed to test the overall hypothesis that the irreversible hypercontracture seen upon reoxygenation is a result of a dynamic and interactive imbalance between cytosolic [Ca2+] and pH, and mitochondrial function. In order to test this hypothesis, the project is organized around the following specific aims. 1) Analyze the relative contributions that the Na+ channel, the Na+/H+ exchanger, and the Na+/K+ pump make to the elevation of [Na+] during anoxia. 2) Test the hypothesis that the reoxygenation-induced Ca2+ spike is caused by the recovery of the Na+/Ca2+ exchanger from inhibition. 3) Determine whether enhanced mitochondrial Ca2+ accumulation early in reoxygenation is beneficial to the cell by significantly lowering cytosolic [Ca2+], or injurious, by causing significant mitochondrial depolarization. 4) Test the hypothesis that the cytosol becomes acidic enough during reoxygenation to retard the onset of hypercontracture, and that the time-course of recovery from acidification is an important factor in determining cell survival. The effects of anoxia on cytosolic [Ca2+], [Na+], and pH will be studied using fluorescent indicators. The effects of anoxia on the Na+/Ca2+ exchanger and the Na+ channel will be studied with the patch-clamp technique. Finally, mitochondrial membrane potential and [Ca2+] will be measured using confocal fluorescence microscopy.